小麦蓝矮病植原体胸苷酸激酶基因(tmk)的分离、原核表达及酶活性分析

Isolation,Prokaryotic Expression and Activity Analysis of Thymidylate Kinase (tmk) Gene from Phytoplasma of Weat Blue Dwarf

【目的】克隆、表达小麦蓝矮病(WBD)植原体胸苷酸激酶基因(tmk),并分析酶活性,进一步研究胸苷酸激酶在植原体感染宿主及繁殖过程中的功能和作用机理,更好地防治植原体病害。【方法】PCR方法扩增tmk基因并进行序列分析,连接pET30a(+)表达载体后原核表达,经Ni-NTA柱层析纯化后进行酶催化活性分析。【结果】首次从小麦蓝矮病(WBD)植原体基因组中分离出胸苷酸激酶基因(tmk),该基因包含tmk-1和tmk-2两种,大小分别为630bp和624bp,其编码的氨基酸序列均包含3个与结合NTP/NMP相关的保守功能区。表达的融合蛋白TMK-1活性极低,酶活仅16.4U/mg,而TMK-2酶活高达112.41U/mg,且其最适催化条件为32℃、pH7.3、1.5mmol/LMg2+和1mmol/LATP。【结论】分析了胸苷酸激酶活性中心的一级结构序列及其催化活性随条件变化而改变的性质,为深入研究小麦蓝矮病植原体胸苷酸激酶在侵染寄主及其在宿主体内增殖的转录性质奠定基础。

[Objective] Wheat blue dwarf （WBD） is an important disease in winter wheat district, which causes serious losses in wheat production. Thymidylate kinase （TMK） catalyses the phosphorylation of dTMP to dTDP in the de novo and salvage pathways of dTTP synthesis in both prokaryotes and eukaryotes. In order effectively control this phytoplasma, we isolated the thymidylate kinase gene of WBD phytoplasma, and analyzed the catalytic activity of TMK protein. [Methods] tmk gene was amplified from the phytoplasma of WBD, the amplicons were digested with EcoR Ⅰ and Hind Ⅲ and then inserted into expression vector pET-30a（＋）. The polyHis-tagged TMK was expressed in E. coli BL21 （DE3） and fusion protein was obtained and purified by Ni-NTA column. The TMK activities were measured by the method of enzyme-coupled assay involving Mg^2＋, dTMP and ATP. [Results] Two genes, tmk-1 and tmk-2 were obtained, with the molecular weight of 630 bp and 624 bp, Both of them encoded an amino acid sequence with three conserved functional motifs which related with binding NTP/NMP. The fusion protein, TMK-2 had a higher catalytic activity （112.41 U/mg） than TMK-1 （16.4 U/mg）, and its optimum catalytic conditions were 32℃, pH7.3, 1.5 mmol/L Mg^2＋ and 1 mmol/L ATP. [Conclusion] TMK-1 and TMK-2 had conserved functional motifs in their primary sequence, and suggested that they may function as TMK enzymes. But, the TMK-1-polyHis fusion protein had very low catalytic activity, the possible reason was that two highly conserved regions were absent in TMK-1, and it might function as another type of kinase in WBD phytoplasma. This experiment lay a foundation for further study of the TMK function in infection and reproduction of WBD phytoplasma.